RESEARCH ARTICLE Mechanisms of nucleotide selection by telomerase Matthew A Schaich1, Samantha L Sanford2, Griffin A Welfer1, Samuel A Johnson2, Thu H Khoang1, Patricia L Opresko2, Bret D Freudenthal1,3* 1Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, United States; 2Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, and UPMC Hillman Cancer Center, Pittsburgh, United States; 3Department of Cancer Biology, University of Kansas Medical Center, Kansas City, United States Abstract Telomerase extends telomere sequences at chromosomal ends to protect genomic DNA. During this process it must select the correct nucleotide from a pool of nucleotides with various sugars and base pairing properties, which is critically important for the proper capping of telomeric sequences by shelterin. Unfortunately, how telomerase selects correct nucleotides is unknown. Here, we determined structures of Tribolium castaneum telomerase reverse transcriptase (TERT) throughout its catalytic cycle and mapped the active site residues responsible for nucleoside selection, metal coordination, triphosphate binding, and RNA template stabilization. We found that TERT inserts a mismatch or ribonucleotide ~1 in 10,000 and ~1 in 14,000 insertion events, respectively. At biological ribonucleotide concentrations, these rates translate to ~40 ribonucleotides inserted per 10 kilobases. Human telomerase assays determined a conserved tyrosine steric gate regulates ribonucleotide insertion into telomeres. Cumulatively, our work provides insight into how telomerase selects the proper nucleotide to maintain telomere integrity. Introduction *For correspondence: During every round of eukaryotic cell division, a small amount of DNA is lost from the ends of each [email protected] chromosome (Olovnikov, 1973; Watson, 1972). Termed the end replication problem, this phenom- enon is countered by two complementary adaptations. First, repetitive noncoding DNA sequences, Competing interests: The known as telomeres, are found at chromosomal ends, preventing the loss of vital genetic information authors declare that no during each cell division (Blackburn and Gall, 1978; Moyzis et al., 1988). Second, the ribonucleo- competing interests exist. protein telomerase elongates shortened telomeres at chromosomal ends using a reverse transcrip- Funding: See page 18 tase activity (Greider and Blackburn, 1987). Without elongation, telomeres will eventually reach a Received: 24 January 2020 critically short length, causing cells to undergo apoptosis or become senescent (Hayflick and Moor- Accepted: 18 May 2020 head, 1961; Meyerson, 1998). Because telomerase plays such a fundamental role in the temporal Published: 05 June 2020 regulation of cell division, aberrations in telomerase are implicated in numerous human diseases. Reviewing editor: Maria Spies, These include premature aging, idiopathic pulmonary fibrosis (IPF), dyskeratosis congenita, and can- University of Iowa, United States cer (Blasco, 2005; Kim et al., 1994; Nelson and Bertuch, 2012). In particular,~90% of cancers upre- gulate telomerase to combat telomere shortening and enable unlimited cell division, as opposed to Copyright Schaich et al. This somatic cells where telomerase is absent (Jafri et al., 2016). article is distributed under the The implication of telomerase in multiple human diseases underscores the importance of under- terms of the Creative Commons Attribution License, which standing its catalytic cycle at the molecular level. Although the human telomerase holoenzyme is permits unrestricted use and composed of multiple accessory subunits, catalysis is localized to the telomerase reverse transcrip- redistribution provided that the tase (TERT) subunit (Nguyen et al., 2018). Historically, biochemical characterization of human TERT original author and source are (hTERT) has proven challenging, in part because of technical difficulties purifying and reconstituting credited. large quantities of active telomerase (Ramakrishnan et al., 1997; Schmidt et al., 2016). As a result, Schaich et al. eLife 2020;9:e55438. DOI: https://doi.org/10.7554/eLife.55438 1 of 21 Research article Biochemistry and Chemical Biology Structural Biology and Molecular Biophysics many fundamental parameters describing the telomerase catalytic cycle have remained undefined. These enzymatic constants are essential for understanding the roles of active site residues, the selec- tion of right from the wrong nucleotides (fidelity), and the prevention of ribonucleotide triphosphate (rNTP) insertion (sugar discrimination). The faithful extension of telomeres by telomerase is critical because aberrations in telomeric sequences prevent shelterin proteins from capping telomeres, thus promoting genomic instability (de Lange, 2005; Nandakumar et al., 2010). Structural studies of human telomerase have also been historically challenging. Currently, the highest resolution structural snapshot of human telomerase is a cryo-EM structure at 8 A˚ resolution (Nguyen et al., 2018). This structure represents a milestone in telomerase structural biology, revealing details of the telomerase tertiary and secondary structure. However, the positions of amino acids are difficult to distinguish at this resolution, leaving many molecular details of the catalytic cycle ambiguous. To mitigate the difficulties inherent in the biochemical characterization of human telomerase, sev- eral model systems have been established. These include models from yeast, the protazoa Tetrahy- mena thermophila (with which a 5 A˚ cryo-EM structure was recently determined), and the insect model Tribolium castaneum (sequence alignment shown in Figure 1—figure supplement 1; Gillis et al., 2008; Jiang et al., 2018; Petrova et al., 2018). For biochemical characterization of TERT, we opted to use T. castaneum TERT (tcTERT) for the following reasons: first, tcTERT readily fit into the cryo-EM density of hTERT and aligns well with the recent cryo-EM structure from T. thermo- phila, highlighting the conserved secondary structure (Figure 1—figure supplement 2); second, upon alignment with hTERT, the active site pocket of tcTERT exhibits a high degree of sequence identity (Supplementary file 1, Table 1a); third, using a truncated version of the T. castaneum telo- merase RNA component (TR), we can readily obtain sufficient quantities of isolated, active tcTERT for characterization of the telomerase catalytic cycle by pre-steady-state kinetics and X-ray crystal- lography (Gillis et al., 2008; Nguyen et al., 2018). Although TERTs have highly conserved active sites, there are significant changes in the domain architecture between human and tcTERT. These include tcTERT lacking the N-terminal (TEN) domain and missing a portion of the insertion in fingers domain (IFD) (Supplementary file 1, Table 1b). These domains are essential for the activity of other telomerase homologs, and have been hypothesized to be particularly important for telomerase ratcheting during translocation (Steczkiewicz et al., 2011). Therefore, we kept our tcTERT kinetics within a single turnover (i.e. insertion) regime, and, wherever possible, complemented the kinetic results with human telomerase studies to characterize the catalytic cycle of telomerase. Using this combined approach, we have elucidated the role of conserved telomerase active site residues and determined the mechanisms of fidelity and rNTP discrimination. Results The TERT subunit of telomerase elongates telomeric DNA using a conserved catalytic cycle as out- lined in Figure 1A, Figure 1—figure supplement 3, and here. First, telomerase anneals its RNA template to the end of telomeric DNA to form a binary complex (TERT:DNA, Figure 1A, state A1). Next, the binary complex binds an incoming dNTP and samples for proper Watson-Crick base pair- ing to the RNA template (Figure 1A, state B1). The transition between these two states represents the nucleotide binding step, measured as a dissociation constant (Kd). If the resulting ternary com- plex (TERT:DNA:dNTP) is in the proper orientation, TERT will catalyze the formation of a phospho- diester bond and extend the telomere by one nucleotide (Figure 1A, state C1). The transition between these two states is the chemistry step, and its theoretical maximum rate with saturating nucleotide concentration is described as kpol. Following insertion of the incoming nucleotide, telo- merase will shift registry to align the active site with the next templating base (forming state A2). This core catalytic cycle repeats six times, until a new telomeric repeat is added (Figure 1A, state C6). All 18 telomerase states that are required to add one telomeric repeat are shown in Figure 1— figure supplement 3 for reference. Importantly, as the telomerase approaches the end of its tem- plate, the DNA:RNA duplex at the 5’ end begins to melt, enabling telomerase to either (1) translo- cate and anneal the RNA component to the newly extended telomeric repeat, thus allowing for additional repeat addition; or (2) dissociate from the telomeric DNA. The number of times that a sin- gle telomerase enzyme traverses this catalytic cycle is tightly regulated. It was recently shown telo- merase becomes inactive after two repeats, but can be reactivated by the recently discovered intracellular telomerase-activating factors (iTAFs) (Sayed et al., 2019). Schaich et al. eLife 2020;9:e55438. DOI: https://doi.org/10.7554/eLife.55438 2 of 21 Research article Biochemistry